In many cases, structures have been determined, often using the lower resolution cryo-electron microscopy (cryo-EM) technique, of picornaviruses in complex with their cellular receptors, neutralizing antibodies, antiviral compounds, or other, biologically significant ligands. Picornavirus capsids are assembled from 60 protomers, each composed of four structural proteins, viral protein 1 (VP1), VP2, VP3, and VP4. The first three of these proteins have molecular weights of around 30 kDa and form the external surface of the icosahedral shell. Conservation of three-dimensional structure is almost invariably greater than conservation of amino acid homology. Thus, structural comparisons can be used to trace divergent evolution over longer time spans than is possible by amino acid sequence comparisons. Assembly of picornaviruses proceeds from 6S protomers of VP1, VP3, and VP0, via 14S pentamers of five 6S protomers, to mature virions. The final step involves inclusion of the RNA into empty capsids or partially assembled shells with simultaneous cleavage of VP0 into VP2 and VP4. The mutant viruses that were able to grow were mostly single mutations and could be sorted into groups that were neutralized by the same set of antibodies. A variety of additional evidence all points to the ability of simple icosahedral viruses to be in constant flux or “breathing”. This unexpected and structurally difficult-to-understand phenomenon accounts for the virus being able to externalize the internal VP4 and amino-terminal region of VP1 in the initial stages of cell entry.

Different virion capsids with icosahedral symmetry. The Τ = 1 shell contains 60 subunits, each represented by a trapezoid that has the approximate shape of the β-barrel. All subunits in the Τ = 1 capsid are identical and are labeled A. The asymmetric unit of the Τ = 3 capsid contains subunits A, B, and C, all of which have the same amino acid sequence but are in slightly different environments. The threefold axis relating A, B, and C is not exact. This quasi-threefold axis also relates the quasi-sixfold axes (left and right Vertexes of the triangle) to a fivefold axis (top vertex). Like the Τ = 1 structure, the Τ = 3 structures are formed by identical subunits with the same β-barrel fold. The Ρ = 3 picornavirus shell, technically a Τ = 1 particle, is closely related to the Τ = 3 shell, being formed by 180 β-barrel domains. The three subunits, labeled VP1, VP2, and VP3, are, however, distinct proteins. The deep, canyon-like depression, the site of receptor attachment in many picornaviruses, is shaded. One 6S protomer assembly intermediate is outlined with a thick black border. The comovirus shell is very similar to the picornavirus capsid, with 180 β-barrels forming the shell. However, there are only two protein types. The large protein (labeled L) is composed of two β-barrel domains (equivalent of VP2 and VP3) covalently linked together. The small subunit (S) is a single β-barrel domain. Reprinted with permission from Rossmann and Johnson (88).

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FIGURE 1

Different virion capsids with icosahedral symmetry. The Τ = 1 shell contains 60 subunits, each represented by a trapezoid that has the approximate shape of the β-barrel. All subunits in the Τ = 1 capsid are identical and are labeled A. The asymmetric unit of the Τ = 3 capsid contains subunits A, B, and C, all of which have the same amino acid sequence but are in slightly different environments. The threefold axis relating A, B, and C is not exact. This quasi-threefold axis also relates the quasi-sixfold axes (left and right Vertexes of the triangle) to a fivefold axis (top vertex). Like the Τ = 1 structure, the Τ = 3 structures are formed by identical subunits with the same β-barrel fold. The Ρ = 3 picornavirus shell, technically a Τ = 1 particle, is closely related to the Τ = 3 shell, being formed by 180 β-barrel domains. The three subunits, labeled VP1, VP2, and VP3, are, however, distinct proteins. The deep, canyon-like depression, the site of receptor attachment in many picornaviruses, is shaded. One 6S protomer assembly intermediate is outlined with a thick black border. The comovirus shell is very similar to the picornavirus capsid, with 180 β-barrels forming the shell. However, there are only two protein types. The large protein (labeled L) is composed of two β-barrel domains (equivalent of VP2 and VP3) covalently linked together. The small subunit (S) is a single β-barrel domain. Reprinted with permission from Rossmann and Johnson (88).

Comparison of the genome organization of CpMV and picornaviruses. RNA2 (left) and RNA1 (right) of CpMV are shown aligned with the RNA of picornaviruses. The molecular weight and function are marked for each gene product. Regions in the two genomes are shaded where the amino acid sequences had been recognized as homologous (4, 31). The 42-kDa structural protein in CpMV (L) contains β-barrel domains that correspond in location to VP2 and VP3 in picornaviruses. The 24-kDa protein in CpMV (S) corresponds to protein VP1 in picornaviruses. The letters C, B, and A indicate the positions occupied by each of these β-barrels in the Τ = 3 quasi-equivalent surface lattice. Reprinted with permission from Rossmann and Johnson (88).

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FIGURE 2

Comparison of the genome organization of CpMV and picornaviruses. RNA2 (left) and RNA1 (right) of CpMV are shown aligned with the RNA of picornaviruses. The molecular weight and function are marked for each gene product. Regions in the two genomes are shaded where the amino acid sequences had been recognized as homologous (4, 31). The 42-kDa structural protein in CpMV (L) contains β-barrel domains that correspond in location to VP2 and VP3 in picornaviruses. The 24-kDa protein in CpMV (S) corresponds to protein VP1 in picornaviruses. The letters C, B, and A indicate the positions occupied by each of these β-barrels in the Τ = 3 quasi-equivalent surface lattice. Reprinted with permission from Rossmann and Johnson (88).

Diagrammatic representation of the polypeptide fold of one subunit of poliovirus found also in the shell-forming portion of most other viral subunit structures. Shown also is the nomenclature for the secondary structural elements βB, βC, . . . , βI. Reprinted with permission from Hogle et al. (51).

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FIGURE 3

Diagrammatic representation of the polypeptide fold of one subunit of poliovirus found also in the shell-forming portion of most other viral subunit structures. Shown also is the nomenclature for the secondary structural elements βB, βC, . . . , βI. Reprinted with permission from Hogle et al. (51).

Diagrammatic representation of the canyon hypothesis. It was suggested that the canyon would be too narrow to allow the binding of a neutralizing antibody. It was also hypothesized that a receptor would be a slender molecule able to bind to the conserved amino acids lining the canyon, thereby avoiding host immune surveillance. The hypothesis was later found to be correct in as far as the site of receptor attachment and shape of the receptor molecule was concerned. However, the footprints of neutralizing antibodies and of the receptor on the viral surface were found to be partially overlapping, which gave some concern as to whether the original hypothesis was based on a correct premise, even though the basic predictions turned out to be correct for rhino- and enteroviruses.

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FIGURE 4

Diagrammatic representation of the canyon hypothesis. It was suggested that the canyon would be too narrow to allow the binding of a neutralizing antibody. It was also hypothesized that a receptor would be a slender molecule able to bind to the conserved amino acids lining the canyon, thereby avoiding host immune surveillance. The hypothesis was later found to be correct in as far as the site of receptor attachment and shape of the receptor molecule was concerned. However, the footprints of neutralizing antibodies and of the receptor on the viral surface were found to be partially overlapping, which gave some concern as to whether the original hypothesis was based on a correct premise, even though the basic predictions turned out to be correct for rhino- and enteroviruses.

Schematic representation of the competition between receptor binding and binding of a pocket factor within the hydrophobic pocket in VP1. The structures represented in gray have been determined crystallographically, and the dotted structure in the top panel has been determined by electron microscopy. When pocket factor binds into the pocket, it deforms the canyon roof, which is also the floor of the canyon. When receptor binds into the canyon, the pocket is presumed to be empty. Thus, the effect of receptor binding is to expel the pocket factor, thereby destabilizing the virus and initiating uncoating, as shown in the top panel. For this to be able to happen, it is necessary for the affinity between the ICAM-1 receptor and the viral surface to exceed that of the antiviral compound (WIN) or pocket factor to the virus. In the middle panel are shown some of the drug-resistant compensation mutations (black spheres) that are on the floor of the canyon. They have been shown to increase the affinity of ICAM-1 for the virus (40). Thus, the binding affinity of ICAM-1 for the mutant virus would now be greater than that for the WIN compounds. Other compensation escape mutations, as shown in the bottom panel, are found lining the hydrophobic binding pocket. These have less bulk than the wild-type residues, thus reducing the affinity of the WIN compounds for the virus. Since the affinity of the receptor ICAM-1 for the virus is unchanged, the equilibrium is altered in favor of receptor binding rather than WIN compound binding. Reprinted with permission from Hadfield et al. (40).

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FIGURE 5

Schematic representation of the competition between receptor binding and binding of a pocket factor within the hydrophobic pocket in VP1. The structures represented in gray have been determined crystallographically, and the dotted structure in the top panel has been determined by electron microscopy. When pocket factor binds into the pocket, it deforms the canyon roof, which is also the floor of the canyon. When receptor binds into the canyon, the pocket is presumed to be empty. Thus, the effect of receptor binding is to expel the pocket factor, thereby destabilizing the virus and initiating uncoating, as shown in the top panel. For this to be able to happen, it is necessary for the affinity between the ICAM-1 receptor and the viral surface to exceed that of the antiviral compound (WIN) or pocket factor to the virus. In the middle panel are shown some of the drug-resistant compensation mutations (black spheres) that are on the floor of the canyon. They have been shown to increase the affinity of ICAM-1 for the virus (40). Thus, the binding affinity of ICAM-1 for the mutant virus would now be greater than that for the WIN compounds. Other compensation escape mutations, as shown in the bottom panel, are found lining the hydrophobic binding pocket. These have less bulk than the wild-type residues, thus reducing the affinity of the WIN compounds for the virus. Since the affinity of the receptor ICAM-1 for the virus is unchanged, the equilibrium is altered in favor of receptor binding rather than WIN compound binding. Reprinted with permission from Hadfield et al. (40).

The pocket factor within the VP1 hydrophobic pocket of coxsackievirus B3 (CVB3) (76). (a) Electron density in the middle of the figure represents the pocket factor in the VP1 pocket, whereas (b) shows the VP1 pocket of CVB3 occupied by antiviral compound WIN 66393. Comparison of the two electron density maps shows clearly that the longer pocket factor has been displaced by the shorter WIN compound. The large peak is the result of an iodine atom in the antiviral compound (76). Reprinted with permission from Muckelbauer et al. (76).

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FIGURE 6

The pocket factor within the VP1 hydrophobic pocket of coxsackievirus B3 (CVB3) (76). (a) Electron density in the middle of the figure represents the pocket factor in the VP1 pocket, whereas (b) shows the VP1 pocket of CVB3 occupied by antiviral compound WIN 66393. Comparison of the two electron density maps shows clearly that the longer pocket factor has been displaced by the shorter WIN compound. The large peak is the result of an iodine atom in the antiviral compound (76). Reprinted with permission from Muckelbauer et al. (76).